Patent EA-003195-B1 (EA)
Title: Method for gene transfer into target cells with retrovirus
Inventor: ASADA KIYOZO
IPC: C12N15/12
Filed: 1996-11-07
Abstract: 1. A method for increasing the efficiency of gene transfer into target cells ex vivo with a retrovirus, comprising the realization of the following steps: (a) pre-stimulating the target cells in presence of target cell growth factor; (b) incubating the retrovirus and the target cells in presence of an artificial substrate; (c) transduction by infecting the target cells with the retrovirus in the presence of a mixture of an effective amount of a functional material having retrovirus binding domain, and an effective amount of another functional material having target cell binding domain. 2. A method according to claim 1, wherein the functional material having retrovirus binding domain is a function material selected from Heparin-II binding region of fibronectin, fibroblast growth factors, collagens, polylysines and functional equivalents thereof. 3. A method according to claim 1, wherein the functional material having target cell binding domain is a ligand. 4. A method according to claim 3, wherein the ligand is selected from cell adhesion proteins, hormones, cytokines, antibodies, carbohydrates. 5. A method according to claim 4, wherein the cell adhesion protein is a cell binding domain polypeptide of fibronectin. 6. A method according to claim 5, wherein the cell binding domain polypeptide of fibronectin is a polypeptide of the binding domain to VLA-5 and/or VLA-4. 7. A method according to claim 4, wherein the ligand is erythropoietin. 8. A method according to any one of claims 1 to 7, wherein the functional materials are immobilized. 9. A method according to any one of claims 8, wherein the functional materials are immobilized on beads. 10. A culture medium for target cells to be used in method for increasing the efficiency of gene transfer into the target cells with a retrovirus according to any of claims 1 to 9 which comprises a mixture of an effective amount of a functional material having retrovirus binding domain, and an effective amount of another functional material having target cell binding domain. 11. A method for localization of a retrovirus-mediated gene transfer into target cells comprising incubating of said retrovirus with a culture medium. 12. A method for localization according to claim 11, wherein the functional material having retrovirus binding domain is a functional material selected from Heparin-II binding region of fibronectin, fibroblast growth factors, collagens, polylysines and functional equivalents thereof. 13. A method for localization according to claim 11, wherein the functional material having target cell binding domain is a ligand. 14. A method for localization according to claim 13, wherein the ligand is selected from cell adhesion proteins, hormones, cytokines, antibodies, carbohydrates. 15. A method for localization according to claim 14, wherein the cell adhesion protein is a cell binding domain polypeptide of fibronectin. 16. A method for localization according to claim 15, wherein the cell binding domain polypeptide of fibronectin is a polypeptide of the binding domain to VLA-5 and/or VLA-4. 17. A method for localization according to claim 14, wherein the ligand is erythropoietin. 18. A method for localization according to any one of claims 11 to 17, wherein the functional materials are immobilized. 19. A method for localization of a retrovirus for gene transfer into target cells which comprises incubating a culture medium according to claim 10 wherein the functional material, and a retrovirus binding domain derived from a fibroblast growth factor, a collagen or a polylysine. 20. A method for localization according to any one of claims 11 to 17, wherein the functional materials are immobilized. 21. A method for increasing the efficiency of gene transfer into target cells ex vivo with a retrovirus, comprising the realization of the following steps: (a) pre-stimulating the target cells in presence of target cell growth factor; (b) incubating the retrovirus and the target cells in presence of an artificial substrate; (c) transduction by infecting the target cells with the retrovirus in the presence of a mixture of an effective amount of a functional material having retrovirus binding domain, and an effective amount of another functional material having target cell binding domain. 22. A method according to claim 21, wherein the functional material having target cell binding domain is a ligand, which specifically binds to the target cells. 23. A method according to claim 22, wherein the ligand is selected from cell adhesion proteins, hormones, cytokines, antibodies, carbohydrates. 24. A method according to claim 23, wherein the cell adhesion protein is a cell binding domain polypeptide of fibronectin. 25. A method according to claim 24, wherein the cell binding domain polypeptide of fibronectin is a polypeptide of the binding domain to VLA-5 and/or VLA-4. 26. A method according to claim 23, wherein the ligand is erythropoietin. 27. A method according to claim 21, wherein the said fibroblast growth factor is selected from a fibroblast growth factor represented by SEQ. ID No. 3 of the Sequence Listing, functional equivalents of the factor and polypeptides containing the factor or functional equivalent of the factor. 28. A method according to claim 21, wherein the functional material is a polypeptide having an amino acid sequence represented by SEQ. ID No. 4 or 5 of the Sequence Listing. 29. A method according to claim 21, wherein the collagen is selected from a fragment having insulin binding domain derived from type V collagen, functional equivalents of the fragment and polypeptides containing the fragment or functional equivalent of the fragment. 30. A method according to claim 29, wherein the fragment having insulin binding domain derived from type V collagen is a fragment having an amino acid sequence represented by SEQ. ID No. 6 of the Sequence Listing. 31. A method according to claim 21, wherein the functional material is a polypeptide having an amino acid sequence represented by SEQ. ID No. 7 or 8 of the Sequence Listing. 32. A method according to any one of claims 21 to 31, wherein the functional material is immobilized. 33. A method according to any one of claims 21 to 31, wherein the functional material is used without immobilization. 34. A culture medium for target cells to be used in method for increasing the efficiency of gene transfer into the target cells with a retrovirus according to any of claims 21 to 33 which can be used in culture medium of target and which contains an effective amount of a functional material derived from a fibroblast growth factor, a collagen or a polylysine, wherein said that each molecula of said material has retrovirus binding domain, and target cell binding domain. 35. A method for localization of a retrovirus, for gene transfer into target cells which comprises incubating of retrovirus with a culture medium according to claim 34. 36. A method for localization according to claim 35, wherein the said fibroblast growth factor is selected from a fibroblast growth factor represented by SEQ. ID No. 3 of the Sequence Listing, functional equivalents of the factor and polypeptides containing the factor or functional equivalent of the factor. 37. A method for localization according to claim 35, wherein the functional material is a polypeptide having an amino acid sequence represented by SEQ. ID No. 4 of the Sequence Listing. 38. A method according to claim 35, wherein the collagen is selected from a fragment having insulin binding domain derived from type V collagen, functional equivalents of the fragment and polypeptides containing the fragment or functional equivalent of the fragment. 39. A method according to claim 38, wherein the fragment having insulin binding domain derived from type V collagen is a fragment having an amino acid sequence represented by SEQ. ID No. 6 of the Sequence Listing. 40. A method according to claim 35, wherein the functional material is a polypeptide having an amino acid sequence represented by SEQ. ID No. 7 or 8 of the Sequence Listing. 41. A method according to any one of claims 35 to 40, wherein the functional material is immobilized. 42. A method according to any one of claims 18,20,32 and 41, wherein the functional material is immobilized on beads. 43. A method for increasing the efficiency of gene transfer into target cells ex vivo with a retrovirus, comprising the realization of the following steps: (a) pre-stimulating the target cells in presence of target cell growth factor; (b) incubating the retrovirus and the target cells in presence of an artificial substrate; (c) transduction by infecting the target cells with the retrovirus in the presence of a mixture of an effective amount of a functional material selected from the group consisting of substantially pure fibronectin, a substantially pure fibronectin fragment or a mixture thereof wherein said that each molecula of said material has retrovirus binding domain, and target cell binding domain. 44. A method according to any one of claims 1 to 9, 21 to 33, and 43, wherein the target cells are cells selected from stem cells, hematopoietic cells, non-adherent low density mononuclear cells, adherent cells, bone marrow cells, hematopoietic stem cells, peripheral blood stem cells, umbilical blood cells, fetal hematopoietic stem sells, embryoplastic stem cells, embryonic cells, primordial germ cell, oocyte, oogonia, ova, spermatocyte, sperm, CD 34 + cells, C-kit + cells, multipotential hematopoietic progenitor cells, unipotential hematopoietic progenitor cells, erythrocyte precursor cells, lymphocytic precursor cells, mature blood cells, lymphocytes, B cells, T cells, fibroblasts, neuroblasts, nerve cells, endothelial cells, angio-endothelial cells, hepatic cells, myoblasts, skeletal muscle cells, smooth muscle cells, cancer cells, myeloma cells and leukemia cells. 45. A method according to any one of claims 1 to 9, 11 to 18, 19 to 33, 35 to 41, and 42 to 44,